Exploring Community Dynamics of
Denitrifying Bacteria in the Cahaba River
Corianne Tatariw, Elise Chapman,
Dr. Jennifer Edmonds
Department of Biological Sciences
University of Alabama, Tuscaloosa


Acidification/
eutrophication of
freshwater systems
Loss of biodiversity
Images from NOAA
•Harmful algal
blooms
•Eutrophication/
hypoxia in coastal
systems
Goal: evaluate denitrification as a
meaningful nitrate sink in large
rivers
How does denitrifier community structure
respond to environmental parameters?
What is the effect of these changes in
denitrifier community structure on rates of
denitrification?
High diversity of
microorganisms
suggests functional
redundancy, but…
Denitrifier communities respond
differently to environmental
conditions, affecting rates of
denitrification (Cavigelli and Robertson 2000)
NO3-
NO2-
NO
nirS /nirK
N2O
N2
gas

Heterotrophic facultative anaerobes

Dissolved Oxygen (DO) affects distribution of nirS and
nirK denitrifiers (Graham et al 2010, Knapp et al 2009)
› nirK denitrifiers can function under fluctuating DO
conditions
› nirS denitrifiers found in consistently anoxic
environments

Measured as
porosity: Fraction of
void space in a
volume of sediment

Greater surface
area: volume in
smaller sediments

Affects movement
of DO through
sediment
VR
FL
CP
Valley Ridge (VR)
Fall Line (FL)
Coastal Plain (CP)
2
P-
C
1
P-
C
FL
-2
FL
-1
-2
VR
-1
VR
Porosity
0.8
0.6
0.4
0.2
-2
-1
P2
C
P1
C
FL
FL
-2
VR
-1
VR
SRP (ppb)
C
C
2
P-
1
P-
2
FL
-
1
FL
-
-2
VR
-1
VR
NO3-N (ppb)
Birmingham
3000
2500
2000
1500
1000
500
0
350
300
250
200
150
100
50
0

NirS and nirK denitrifiers will have distinct
community structures within each
geophysical province due to changes in
sediment size and channel slope.

Dominance of nirS vs. nirK will vary due to
spatial and seasonal variations in DO

Three subsamples taken from homogenized
sediment in field
› Spring, Early Summer, Late Summer, Winter
› 2 locations per geophysical province (VR, FL, CP)

TRFLP of nirS and nirK
› nirS: cd3aF/R3cd-HEX
› nirK: FlaCu-FAM/R3Cu
› Restriction enzyme: MspI

Analysis of Similarity: Primer v.6
2D Graph 2

No significant
difference by
geophysical
province or
date

nirS dominates
in anoxic
environments
which may
have less
variable
conditions
2D Stress = 0.14
Valley Ridge
X Fall
vs Y - CP
Line
X vs Y - FL
Coastal Plain
X vs Y - VR
Global R = 0.157
p = 0.03
nirS

Coastal Plain
different from
Valley Ridge
and Fall Line

No significant
seasonal
difference

CP has finer,
sandy substrate
compared to VR
and FL
2D Stress = 0.22
Valley
Coastal
PlainRidge
Fall
LineLine
Fall
Valley Ridge
Coastal Plain
Global R = 0.223
p = 0.001

Denitrification rates will vary among
geophysical provinces in response to
changes in denitrifier community
composition.

Highest rates of denitrification will
coincide with high primary productivity
as carbon limitation is alleviated

Sediment samples collected with
microbial samples

Acetylene block method to measure
denitrification potential rates
5
0
5
0
5
0
Denitrification Rate (mmol N2 g-1 hr-1)
Denitrification Potential Rate (nmol/g/hr)
0
p <0.05
b
30
25
ab
20
ab
15
10
a
5
VR
FL
CP
0
Spring
Early Summer
Late Summer
Winter
VR
Apr Amb
Jun Amb
Sept Amb
FL
CP
The Cahaba River is
a sink for NO3-
Lack of relationship
between changes in
community composition
and denitrification rates
suggests functional
redundancy
Committee Members
Dr. Jennifer Edmonds
Dr. Robert Findlay
Dr. Lisa Davis
Lab Members
Elise Chapman
Courtney Dragiff
Susan Jozefiak
Ben Wilson
Field and Lab Help
Chau Tran
Julie Jarnigan
Diane Schneider
Marie Wilson
2D Stress = 0.16
• Bacterial
community
structure
changes
over time
through
macrophyte
growing
season
2 June 2010
14 July 2010
24 June 2010
2 June 2010
2 June
20102010
24 June
24 June 2010
14 July 2010
14 July 2010
Global R =
0.414
p = 0.001
VR
DNP (nmol N2 g-1 hr1)
20
*
Dec.
2010
Ambient
500 M N
500 M N/ 2500 M C
15
10
16
5
14
0
VR
12
FL
CP
10
6
4
2
0
VR
* Statistically significantly different from
ambient denitrification potential rate; p=0.05
DNP (nmol N2 g-1 hr1)
8
Apr. 2011
FL
CP
Fall Line

Estimated deposition of
reactive nitrogen in a)
1890s and b) 1990s
• Fertilizer
• Leguminous
crops
• Livestock
• Fossil fuel
combustion
Galloway et al. 2004. Biogeochemistry 70:
Burgin and Hamilton. 2007. Front Ecol. Environ. 5: 8996
nirS: Consistently anoxic
environments, always
active
nirK: Areas of DO
flux, more active at
night
Nitrite reductase cytochrome-cd1
Copper-containing nitrite reductase
Nurizzo et al. 1998. Biochemistry 37: 1398713996.
Nojiri et al. 2007. PNAS. 104: 4315-4320.
Denitrification is occurring along the
Cahaba at rates comparable to those in
literature (Piña-Ochoa and ÁlvarezCobelas 2006)
 Highest rates occurred at the Fall Line in
summer

› Lack of C response suggests in-stream
primary producers alleviated C-limitation
› Spatial heterogeneity at Fall Line
Ambient
+N (500 M)
+N+C (500/2500 M)
(nmol(nmol
N2O/g/hr)
Potential
DenitrificationDNP
N2 g-1
hr-1)
50
40
30
20
10
0
VR1
VR2
FL1
FL2
Site
CP1
CP2
2D Graph 5
Ambient
+N (500 M)
+N+C (500/2500 M)
**
60
DNP (nmol N2O/g/hr)
50
40
30
20
*
*
*
*
10
0
VR1
VR2
FL1
FL2
Site
*
Significantly different from ambient DNP rate;
p <0.05
CP1
CP2
1) Denitrification
potential
rate vary in the
How do rates of
denitrification
measurements
Cahaba River? in April 2011 will be highest
at
the Fall
Line
2) How
does
denitrifier community
composition
change
over
space
and
time?
2) T-RFLP of nirS and nirK functional genes
will show distinct denitrifier community
structure at the scale of geophysical
3)
How
qPCRdoes
of nirS
denitrifier
and nirKactivity
will show
and
increased
province.
abundance
denitrifier
activity
change
andover
abundance
space and
time?
associated
with higher denitrification rates
and there will be changes in nirS vs. nirK
activity over space and time.
Date
Site
Jun 2010 LB
HL
CV
SP
MJ
03 Jun 2010 PP
24 Jun 2010 PP
14 Jul 2010 US
MB
SC
DD
Aug 2010 PP
Sep 2010 PP
Sep 2010 LB
HL
CV
SP
MJ
Dec 14 2010 LB
HL
PP
CV
SP
MJ
Avg Ambient StDev
Temperature °C
8.161998949 0.332226
29.2
6.289706525 0.641923
29.5
3.162459464 0.686203
29
4.703626103 3.821566
29
4.994243446 0.058324
18.83327397 7.527388
28.9
26.41588141 3.552288
30.1
6.471659572 1.451805
30.7
4.279251108 1.649617
4.420255345 1.476759
4.165626458 1.037914
8.372572675 3.651942
32.2
11.56170254 12.80802
28.8
5.767593586 1.34986
7.111404042 0.805633
10.28448042 4.559856
5.749148825 0.785916
1.982245124 0.201137
8.917669919 2.055431
4.5
6.862733321 1.916889
6
18.48504912 2.247254
6.1
6.692058788 1.784798
6.893814842 6.023206
6.7
7.50694151 0.692246
6.5
Porosity
0.386133333
0.373494737
0.3837
0.357856642
0.392133333
StDev
0.006886
0.018102
0.014843
0.009515
0.020305
AFDM
2.601236945
2.283198858
1.740152112
0.386379651
0.633802713
StDev
0.377843
0.254031
0.08738
0.085188
0.624277
0.478411111 0.057839
3.764592511 1.411641
0.38225
0.38875
0.382
0.3175
0.37925
0.286
0.2612
0.219333333
0.401066667
0.588266667
0.682933333
3.987856567
2.071134073
1.559518416
0.281233063
0.410048422
3.806077359
2.829575575
4.112442571
2.034080692
3.732548565
3.302282683
0.00916
0.365
0.007104
0.00952
0.030766
0.069527
0.056908
0.032934
0.028381
0.019129
0.026544
0.847384
0.239217
0.12772
0.199009
0.369577
0.235186
0.736192
0.405341
0.6658
1.639047
0.318121
•Large and unregulated
•North of Birmingham to
Selma, AL
•Confluence with
Alabama River
•Valley Ridge, Fall Line,
Coastal Plain
How do rates of denitrification vary in
Denitrification
streams receiving large
the Cahaba in
River?
1)
inputs of NO3 is limited by organic
carbon lability.
2)
How
does
denitrifier
community
• Primary producer distribution will
composition
change over
space and
control
denitrification
longitudinally.
time?
• Seasonal changes in labile OC
availability will affect rates of
3)denitrification.
How does denitrifier activity and
abundance change over space and
time?
5
0
100
200
(nmol N2 g-1 hr-1)
Potential
Denitrification
DNP (nmol
N20/g/hr)
[
CP-2
December
2010
Coastal
Plain
Site 2 – Dec
2010
No Carbon
Carbon
20
15
10
5
0
100
200
300
400
NO3- Concentration
(mmol)
[NO3-Added
]
No Carbon
Carbon
500
No Carbon
Carbon
FL-2 December 2010
Fall Line Site 2 – Dec 2010
DNP (nmol
N20/g/hr)
(nmol N2 g-1
Potential
Denitrification
hr-1)
12
9
6
3
0
100
200
300
400
[NO3-] Added (mmol)
NO3- Concentration
500
› -Delivery of NO3- to subsurface influenced by
geomorphology (Böhlke et al. 2009)
› -Competition with in-stream primary producers
Early
*
Summer
VR
Fall Line Late
Summer
DNP (nmol N2 g-1 hr1)
60
40
0
*
50
30
FL
Ambient
500 M N
500 M N/ 2500 M C
40
20
30
20
10
No significant response to
nitrogen or carbon
(p=0.05)
10
00
VR
FL1
FL
FL2 CP
X Data
FL1: Middle of Fall Line,
macrophyte-dominated
shoals
50
Late Summer
DNP (nmol N2 g-1 hr1)
Col 27
Col 29
Col 31
40
30
F2: Bottom of Fall Line,
transition to Coastal Plain
* Statistically significantly different from
ambient denitrification potential rate; p=0.01,
0.02
20
10
0
VR
FL
CP